Persistence of a herpesvirus - in both the host and population - requires a balance between latent and lytic phases. This balance is also important for herpesvirus-mediated pathogenesis, including EBV-related diseases. EBV provides a powerful system for studying human herpesvirus latency (and lytic activation), due to its ability to establish latency in vitro. While latent EBV can be (re-)activated into the lytic phase by chemicals or immunoglobulin crosslinking, not every EBV-infected B cell is susceptible to lytic activation. This refractory state, while crucial for viral persistence, negatively impacts success f viral oncolytic therapy for EBV-cancers. Unfortunately, determinants of susceptibility to lytic activation signals are poorly understood. To address this critical issue at the single cell level, e pioneered a method for separating lytic and refractory B cells, and made the fundamental discovery that cellular STAT3, a transcription factor overactive in many human cancers, regulates susceptibility to lytic activation. We propose to test the hypothesis that STAT3 does this by employing cellular DNA-binding protein SZF1 to recruit the transcriptional co-repressor KAP1 to simultaneously repress multiple EBV lytic genes, thereby promoting the refractory state. In Aim 1, we will identify and validate SZF1-binding sites on the EBV genome in infected cells, using ChIP-exo to achieve single nucleotide resolution. In Aim 2, we will elucidate the mechanism of SZF1-mediated transcriptional repression of EBV lytic genes - using STAT3, SZF1, and KAP1 mutants, as well as purified lytic and refractory cells. The proposed research is expected to significantly impact our understanding of 1) how 2 major host transcriptional mechanisms (mediated via STAT3 and KAP1) regulate expression of EBV lytic genes to affect susceptibility to lytic activation, and thereby viral persistence, 2) how STAT3, SZF1, and KAP1 function may be exogenously manipulated to increase the number of lytic cells, and 3) how KAP1 repressor identifies targets in a genomic context - promoting discovery of cellular targets and physiologic functions of SZF1. Importantly, by providing tools to devise approaches (e.g., small molecules) to enhance lytic activation, our work promises to cripple viral persistence, and improve effectiveness of therapies for EBV-associated cancers.